Innate immunity is the first line of defense against pathogens and is highly conserved from insects to humans. While many key facets of the innate immune system have been elucidated, there is a fundamental gap in our knowledge of the pathways that restrict arthropod-borne viral infections both in the insect vector and the mammalian host. Importantly, a molecular understanding of these mechanisms is essential to overcome the lack of effective antiviral therapeutics and combat human disease. The Drosophila immune response is highly homologous to that of vector insects and additionally shares striking similarities with mammalian innate immunity. Thus, discoveries in fruit flies may have profound impacts on human health by identifying novel approaches to interfere with disease transmission in vector species and revealing conserved immune pathways in mammals. One essential pathway that restricts the human arbovirus Vesicular Stomatitis virus (VSV) in Drosophila is autophagy, an ancient intracellular degradative program evolutionarily conserved from yeast to humans. Based on preliminary data, this response depends on the interaction between VSV and a previously uncharacterized Toll receptor, Toll-7; however, the downstream pathways and global significance of Toll receptors remain unknown. The long-term objective of this application is to define the molecular mechanisms by which Drosophila recognizes viral infections to induce protective antiviral autophagy. Because these pathways share a number of molecular and functional similarities to mammalian TLR pathways, and increasing in vitro data suggest that TLRs activate autophagy during a variety of infections, the proposed research also has the potential to extend our understanding of the mechanisms that link innate immunity to autophagy in mammals. As such, this application proposes three specific aims: (1) elucidate the mechanism by which virus is targeted by autophagy; (2) identify the role of Toll receptors in antiviral autophagy and defense; and (3) explore the signaling pathways that link virus recognition to autophagy in flies and mammals. These studies will expand our understanding of the molecular mechanisms underlying antiviral autophagy in flies, as well as conserved autophagy-inducing pathways likely downstream of TLR signaling in mammals. Moreover, this research opens a previously unappreciated and unexplored realm of Toll receptors in immunity, which may serve as effective targets for novel vector-based antiviral therapeutics given their conservation in other insects.